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T Kealey

Publications and source records attributed to T Kealey.

At least 19 recordsLinked to original sources
PubMed2006

An E box in the exon 1 promoter regulates insulin-like growth factor-I expression in differentiating muscle cells.

Insulin-like growth factor (IGF)-I expression is subject to complex temporal and spatial regulation. Endocrine synthesis occurs in the liver, where transcription is initiated from promoters located in either exon 1 (P1) or in exon 2 (P2), whereas local transcription is mainly initiated from P1. IGF-I is expressed in a range of tissues and, in particular, is an important regulator of skeletal muscle mass, although the mechanisms of tissue-specific regulation remain to be fully characterized. Gene regulation in skeletal muscle is associated with the E box DNA element (5'-CANNTG-3') recognized by myogenic regulatory factors (MRFs), such as MyoD1. Transcription element profiling identified a hypothetical myogenic E box (sequence 5'-CAGCTG-3') within P1, immediately upstream of the major muscle transcriptional start site, and we sought to test its activity in differentiating C2C12 myoblasts. We found P1-driven IGF-I mRNA expression to be associated with myogenic differentiation and, moreover, that a single base-pair mutation in the E box specifically reduced expression in myofibers. A synthetic enhancer construct containing a triplet repeat of the E box was active in muscle cells and strongly induced in myofibers. The capacity of a double-stranded IGF-I E box probe (but not one bearing a single-base pair alteration) to bind C2C12 nuclear lysates increased with myogenesis, and a transactivation assay demonstrated that the E box was recognized by E protein-MRF heterodimers. Mechanisms of tissue-specific gene activation are of increasing biological interest, and we have identified a cis-element able to direct muscle-specific IGF-I gene expression.

Animals↗
PubMed2005

Gene expression profiling of the ageing rat vibrissa follicle.

BACKGROUND: The application of gene expression profiling to the study of chronological ageing has the potential to illuminate the molecular mechanisms underlying a complex and active process. For example, ageing of the skin and its constituent organs has myriad phenotypic consequences, and a better understanding of the means by which these changes arise has important corollaries for intervention strategies. OBJECTIVES: We used a transcriptional profiling approach to investigate changes in gene expression associated with ageing of the large vibrissa follicle of the Wistar rat. METHODS: Follicle mRNA isolated from male Wistar rats at 1 and 18 months of age was hybridized to Clontech Atlas 1.2 Rat cDNA macroarrays. Confirmation of array results was provided by the use of Northern blotting and immunohistochemistry. RESULTS: Seven transcripts displayed at least a 1.6-fold increase in expression with age, of which APOD (2.5-fold), GSTM2 (2.0-fold) and NPY (1.8-fold) showed the greatest increases. Decreased expression was found in 19 transcripts, most notably in ALOX12 (13.3-fold) and GAP43 (12.6-fold) expression. CONCLUSIONS: Follicular ageing is characterized by transcriptional changes associated with diverse aspects of keratinocyte metabolism, proliferation and development.

Aging↗
PubMed2004

Human sebaceous glands engage in aerobic glycolysis and glutaminolysis.

BACKGROUND: The skin and its appendages support aerobic glycolytic and glutaminolytic metabolism. Their major fuels are glucose and glutamine, which are, however, largely catabolized anaerobically. OBJECTIVES: For the human sebaceous gland it has been reported that glucose, lactate and acetate provide good lipogenic substrates but that glutamine does not. Therefore, we have investigated the intermediary metabolism in vitro of freshly isolated human sebaceous glands to determine if their metabolism of glutamine is anomalous relative to the rest of the skin. METHODS: Glycolytic rate, glucose and glutamine oxidation, and glucose metabolism by the pentose phosphate pathway were determined in freshly isolated human chest sebaceous glands. Further, sebaceous intermediary metabolites were analysed using spectrophotometry and high-performance liquid chromatography. Moreover, glands were maintained in vitro as whole organs to investigate the effects of precursors and inhibitors of polyamine synthesis on rates and patterns of lipogenesis and DNA synthesis. RESULTS: We confirm that the human sebaceous gland is a glycolytic and glutaminolytic tissue. Glucose is mainly converted to lactate, with only 6% of glucose being oxidized to CO(2). Glutamine is largely converted to glutamate, alanine, serine, glycine, aspartate, threonine, lactate and ammonia, with only 12% being oxidized. We have also shown that exogenous glutamine is required for cellular proliferation and lipogenesis by human sebaceous glands. However, in its absence spermidine could fully restore rates of DNA synthesis and lipogenesis. CONCLUSIONS: Although glutamine is a poor substrate for sebaceous lipogenesis, this cannot be attributed to its lack of catabolism. We have shown that glutamine is an essential fuel, but that it can be replaced by exogenous spermidine. Therefore, we suggest that in sebocytes both glutamine and spermidine may act as essential purine and pyrimidine precursors.

Aerobiosis↗
PubMed2004

Peroxisome proliferator-activated receptor and farnesoid X receptor ligands differentially regulate sebaceous differentiation in human sebaceous gland organ cultures in vitro.

BACKGROUND: Nuclear hormone receptors are important in the regulation of epidermal differentiation and have been implicated in lipid metabolism. In particular, there is evidence suggesting that the activation of peroxisome proliferator-activated receptors (PPARs) is an important factor in the regulation of sebocyte lipogenesis. OBJECTIVES: To determine the role of PPARs, farnesoid X receptor (FXR) and other orphan nuclear hormone receptors in sebaceous gland function in vitro by investigating the biochemical effects of appropriate ligands, and by establishing the RNA and protein expression patterns of a number of nuclear receptors in sebaceous glands ex vivo. METHODS: Human chest sebaceous glands were maintained in vitro as freshly isolated and as 7-day cultured whole organs. We then studied the effects of appropriate ligands on the glandular rates of lipogenesis and DNA synthesis, as well as determining the mRNA (reverse transcription-polymerase chain reaction) and protein expression patterns (immunohistochemistry/immunoblotting) of the nuclear hormone receptors of interest. RESULTS: PPAR ligands, but not FXR ligands, inhibited sebaceous lipogenesis, in particular the PPARalpha ligands LY 171883 and WY 14643, and the PPARgamma ligands BRL 49653 and 15-deoxy-Delta-12,14-prostaglandin J(2). We detected RNA expression of PPARalpha, PPARbeta, PPARgamma, retinoid X receptor alpha, liver X receptor alpha (LXRalpha) and pregnane X receptor but not FXR in freshly isolated and 7-day maintained sebaceous glands. PPARalpha, PPARbeta, PPARgamma and LXRalpha protein were detected in nuclear extracts of sebaceous glands. CONCLUSIONS: We conclude that activation of nuclear hormone receptors, in particular activation of PPARalpha and PPARgamma, can regulate lipogenesis in human sebaceous glands. As suppression of sebum secretion is associated with reduced acne activity, the nuclear hormone receptors involved may open new avenues in the development of novel acne treatments.

Cell Differentiation↗
PubMed2002

Modelling the remission of individual acne lesions in vitro.

BACKGROUND: Acne lesions spontaneously remit, but the mechanism of this remission has not been elaborated. It is known, however, that the remission is associated with a de-differentiation of sebocytes, causing a cessation of sebum secretion specific to that particular pilosebaceous unit. We have previously described the cytokines that will promote in vitro the lesions of acne. OBJECTIVES: To show that those same cytokines may also promote a de-differentiation of sebocytes analogous to that seen during remission of some lesions. METHODS: Human chest sebaceous glands were maintained in vitro as whole organs. We then chronicled the effects of the appropriate cytokines and growth factors on the glandular rates of (i) lipogenesis and (ii) DNA synthesis, as well as on (iii) glandular morphology, (iv) the expression patterns of the proliferation marker Ki-67, (v) keratinocyte-specific markers, and (vi) the sebocyte marker epithelial membrane antigen. RESULTS: We have shown that the same cytokines that promote comedogenesis (interleukin-1alpha), expression of infundibular intercellular adhesion molecule-1 and human leucocyte-associated antigen-DR (tumour necrosis factor-alpha and interferon-gamma), and infundibular disruption (epidermal growth factor/transforming growth factor-alpha) in human infundibula in vitro, will also inhibit sebaceous lipogenesis in vitro and will also induce, histologically, a de-differentiation of human sebocytes into a keratinocyte-like phenotype. CONCLUSIONS: These results confirm our hypothesis that the cytokines that induce the infundibular changes in acne may also inhibit the secretion of lipid from the sebaceous gland and thus, on diffusing down to the gland, contribute to the remission of the individual lesions. These findings help to explain the known natural history of the disease.

Acne Vulgaris↗
PubMed2001

Evidence for sodium-coupled acid-base transport across the basolateral membrane of the reabsorptive duct of the human eccrine sweat gland.

Intracellular pH was measured in isolated nonperfused ducts of human eccrine sweat glands in vitro to investigate basolateral acid-base transport mechanisms. Bath sodium removal led to a bicarbonate-independent, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid insensitive acidification. The recovery of this acidification was ethylisopropyl amiloride sensitive, suggestive of basolateral sodium:hydrogen exchange. Whereas bath chloride removal led to a small acidification this was not 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive and its causes remain unclear. Elevation of bath potassium to depolarize the basolateral membrane led to a small alkalinization but this was not mimicked by addition of barium or chloride removal. As chloride removal and barium addition would be expected to cause larger depolarizations than potassium elevation these observations do not support a major role for electrogenic acid-base transport. In conclusion, although this study does not support a major role for electrogenic acid-base transport, it has demonstrated the basolateral presence of sodium-coupled acid-base transport in the reabsorptive duct of the human eccrine sweat gland, which most likely represents a sodium:hydrogen exchanger involved in regulation of intracellular pH.

Absorption↗
PubMed2000

Id proteins are dynamically expressed in normal epidermis and dysregulated in squamous cell carcinoma.

Helix-loop-helix genes regulate many developmental pathways, and growing evidence associates dysregulated expression with tumorigenesis. We observed Id-1, Id-2, and Id-3 mRNA expression in proliferating human keratinocytes in vitro with subsequent down-regulation with differentiation. Immunohistochemical analysis of human tissue sections identified cytoplasmic Id-1 expression and nuclear Id-2 and Id-3 expression in the proliferating layers of the epidermis. Furthermore, we observed a columnar pattern of Id-2 and Id-3 staining, which may relate to the epidermal proliferative unit. In squamous cell carcinoma of the head and neck, Id protein immunoreactivity was observed in the majority of malignant keratinocytes in the most poorly differentiated sections, with reduced staining in well-differentiated disease.

Carcinoma, Squamous Cell↗
PubMed2000

Cyclical changes in rat vibrissa follicles maintained In vitro.

In mammals hair growth is cyclical; however, the factors that regulate the hair growth cycle are still poorly understood. The recent development of methods for culturing hair follicles in vitro has proved an important tool to investigate many aspects of the regulation of hair follicle growth. At present, however, these models are based on the culture of anagen hair follicles and have only partially been used to address the cyclical nature of hair growth. In this study we have made use of the fact that in rodents the hair growth cycle is synchronized, well characterized, and relatively short. We have isolated vibrissa follicles from 12 d old rats and confirmed by histology that these follicles are in the anagen stage of their first hair growth cycle. We have then maintained these follicles in vitro, on Gelfoam supports, for up to 23 d (35 d of age) and compared their histology with in vivo follicles from equivalent age littermates. We observed that 12 d old follicles maintained in vitro for up to 23 d show changes in morphology that suggest that cultured rat vibrissa follicles retain cyclical activity in vitro. Cyclical changes in hair follicle morphology were only seen in follicles maintained on gelfoam supports and moreover, hair follicle size appears to be a key feature in determining the ability of the follicle to cycle in vitro. All follicles that showed cyclical changes in vitro, however, appeared to remain blocked in pro-anagen. These data suggest that the vibrissa follicle is a in vitro good model system with which to investigate hair cycle control. J Invest Dermatol 115:1152-1155 2000

Animals↗
PubMed1998

The effects of inflammatory cytokines on the isolated human sebaceous infundibulum.

The human sebaceous pilosebaceous infundibulum was isolated and maintained in medium for up to 7 d. Freshly isolated infundibula were found to express keratins 1, 5, 6, 16, and 17, as determined by immunohistochemistry. In addition, freshly isolated infundibula expressed filaggrin, profilaggrin, involucrin, cornifin alpha, and loricrin. This pattern of expression was retained over 7 d. The addition of 100 U interferon (IFN)-gamma per ml over 3 d and 1 nM phorbol myristate acetate over 24 h resulted in the expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR by infundibular keratinocytes, as determined by immunohistochemistry. Ten nanograms tumor necrosis factor-alpha per ml and 10 ng IL-6 per ml both caused expression of ICAM-1 alone. IL-1alpha had no effect on the expression of ICAM-1 or HLA-DR over 3 d, but addition of 1 ng IL-1alpha per ml over 7 d in culture resulted in hypercornification of the keratinocytes of the infundibulum, apparently brought about by early keratinocyte cornification. These data suggest that the isolated, maintained, infundibulum is a good model for studying the effects of inflammatory cytokines on the infundibulum, and that IL-1alpha acts on infundibular keratinocytes to promote cornification.

Cell Differentiation↗
PubMed1998

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol.

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

Acetyl-CoA Carboxylase↗
PubMed1998

Lipogenesis in the human sebaceous gland: glycogen and glycerophosphate are substrates for the synthesis of sebum lipids.

The lipid class compositions of freshly isolated and overnight maintained human sebaceous glands were determined using high performance thin layer chromatography and were found to be broadly similar. The lipid classes identified in freshly isolated glands were broadly similar to those generated from various radiolabeled precursors in vitro, although lower amounts of wax/sterol esters and cholesterol were observed in vitro. We determined the glycogen content of sebaceous glands and showed that during their incubation with several radiolabeled substrates, with the exception of [U-14C]glucose, significant glycogen breakdown occurred, thus providing acetyl-CoA and NADPH that could affect the pattern of lipids synthesized. We examined glycogen-depleted glands and found that their rates and pattern of lipogenesis resemble that of nondepleted glands, except that the squalene/triacylglycerides ratio for acetate and glutamine doubled from 1.6:1 to 3.4:1 and from 0.7:1 to 1.4:1, respectively. We have shown that exogenous glycerol reduces the squalene/triacylglycerides ratio from acetate from 3.4:1 to 1.7:1, suggesting that glycogen-derived glycerophosphate is important in triacylglycerides synthesis from acetate. Moreover, glands express a glycerokinase activity that may fully account for the rate of triacylglycerides synthesis seen in vitro. We conclude that glycerophosphate generation may be as important as NADPH generation in explaining the directing effects of different substrates within the sebaceous gland.

Female↗
PubMed1998

The organ-maintained human sebaceous gland.

In the absence of epidermal growth factor (EGF) and phenol red in the maintenance medium, human sebaceous glands apparently retain in vivo rates of cell division and lipogenesis over 7 days and also retain in situ morphology. This is reversible on the re-addition of 10 ng/ml EGF and 10 mg/ml phenol red. The addition of 600 pM 17 beta-oestradiol results in a significant fall in the rate of lipogenesis over 7 days' maintenance, without affecting the rate of cell division. This effect is apparently due to abnormal differentiation of newly formed sebocytes. Neither 1 nM testosterone nor 1 nM dihydrotestosterone has any effect on rates of cell division or lipogenesis over 7 days. 1 microM 13-cis-retinoic acid causes a significant reduction in the rate of lipogenesis over 7 days.

Adult↗
PubMed1998

Modelling the infundibulum in acne.

The human sebaceous-pilosebaceous infundibulum was isolated and maintained for 7 days. Infundibular viability was maintained over 7 days in this medium. The addition of 1 ng/ml interleukin 1 alpha (IL-1 alpha) caused hypercornification of the infundibulum similar to that seen in comedones which could be blocked by 1,000 ng/ml IL-1 receptor antagonist. However, in contrast to interferon gamma 1, IL-1 alpha had no effect on the expression of intercellular adhesion molecule or human leucocyte antigen type DR in infundibular keratinocytes. The addition of 5 ng/ml epidermal growth factor or 5 ng/ml transforming growth factor alpha to the medium caused a disorganization of the keratinocytes of the infundibulum. We are now, therefore, able to model histologically the major infundibular changes in acne. Furthermore, we suggest that IL-1 alpha causes the scaling seen in many inflammatory skin diseases.

Antigens, CD↗
PubMed1997

The regulatory biology of the human pilosebaceous unit.

The last few years have witnessed an acceleration in our understanding of the regulation of the human pilosebaceous unit. Recombination and histochemical experiments are beginning to elucidate the role of homeotic genes, transcription factors, growth factors and adhesion molecules in pilosebaceous embryology. Histochemical studies, experiments in gene-modified animals, and in vitro studies on growing human hairs, have identified a number of growth factors that are central to normal hair growth. Thus epidermal growth factor and transforming growth factor-alpha appear to be involved in the triggering of both anagen and catagen. Insulin-like growth factor-I appears to sustain normal anagen growth, transforming growth factor-beta will inhibit anagen growth, while interleukin-1-alpha and tumour necrosis factor-alpha will induce matrix cell death. These complex growth factor effects are beginning to be moulded into an integrated model of pilosebaceous regulation. The role of steroid hormones in modulating these growth factor effects is also beginning to be understood.

Cytokines↗